Independent manipulation of histone H3 modifications in … · 2020. 4. 30. · bivalent nucleosome system that allowed for the constitutive assembly of asymmetrically modified sister

*For correspondence: jqzhou@

sibcb.ac.cn

†These authors contributed

equally to this work

Competing interests: The

authors declare that no

competing interests exist.

Funding: See page 20

Received: 05 July 2017

Accepted: 12 October 2017

Published: 13 October 2017

Reviewing editor: Tim Formosa,

University of Utah School of

Medicine, United States

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Independent manipulation of histone H3modifications in individual nucleosomesreveals the contributions of sisterhistones to transcriptionZhen Zhou1†, Yu-Ting Liu1†, Li Ma2, Ting Gong1, Ya-Nan Hu2, Hong-Tao Li1,Chen Cai1,3, Ling-Li Zhang1, Gang Wei2, Jin-Qiu Zhou1,3*

1State Key Laboratory of Molecular Biology, CAS Center for Excellence in MolecularCell Science, Innovation Center for Cell Signaling Network, Shanghai Institute ofBiochemistry and Cell Biology, University of Chinese Academy of Sciences, ChineseAcademy of Sciences, Shanghai, China; 2Key Laboratory of Computational Biology,CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes forBiological Sciences, Chinese Academy of Sciences, Shanghai, China; 3School of LifeScience and Technology, Shanghai Tech University, Shanghai, China

Abstract Histone tail modifications can greatly influence chromatin-associated processes.

Asymmetrically modified nucleosomes exist in multiple cell types, but whether modifications on

both sister histones contribute equally to chromatin dynamics remains elusive. Here, we devised a

bivalent nucleosome system that allowed for the constitutive assembly of asymmetrically modified

sister histone H3s in nucleosomes in Saccharomyces cerevisiae. The sister H3K36 methylations

independently affected cryptic transcription in gene coding regions, whereas sister H3K79

methylation had cooperative effects on gene silencing near telomeres. H3K4 methylation on sister

histones played an independent role in suppressing the recruitment of Gal4 activator to the GAL1

promoter and in inhibiting GAL1 transcription. Under starvation stress, sister H3K4 methylations

acted cooperatively, independently or redundantly to regulate transcription. Thus, we provide a

unique tool for comparing symmetrical and asymmetrical modifications of sister histone H3s in vivo.

DOI: https://doi.org/10.7554/eLife.30178.001

IntroductionIn eukaryotes, chromatin carries both genetic and epigenetic information that controls multiple cellu-

lar processes, such as DNA replication, transcription and genome organization (Berger, 2007;

Lawrence et al., 2016; Papamichos-Chronakis and Peterson, 2013). The basic unit of chromatin is

the nucleosome, which comprises ~147 bp of DNA and a histone octamer formed by two copies of

histone H2A-H2B and H3-H4 heterodimers (Bentley et al., 1984; Kornberg and Thomas, 1974;

Luger et al., 1997; Oudet et al., 1975). The packaging of DNA into nucleosomes affects sequence

accessibility, and nucleosomes therefore regulate the activity of DNA-binding proteins (Lee et al.,

1993; Wasylyk and Chambon, 1979). Histones also appear to protect DNA from breaking and

maintain the fidelity of both replication and transcription (Carrozza et al., 2005; Govind et al.,

2007; Joshi and Struhl, 2005; Keogh et al., 2005; Pinskaya et al., 2009). The regulation of nucleic

acid metabolism by nucleosomes is mediated through multiple post-translational modifications

(PTMs), such as methylation, acetylation, phosphorylation, and sumoylation (Lawrence et al., 2016).

Histone lysine methylation, especially on histone H3, regulates chromatin structure and transcrip-

tion (Ng et al., 2002; Vermeulen and Timmers, 2010; Wagner and Carpenter, 2012). In budding

Zhou et al. eLife 2017;6:e30178. DOI: https://doi.org/10.7554/eLife.30178 1 of 24

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yeast, the best-studied methylations on histone H3 are methylation of lysine at amino acid positions

4, 36, and 79 (H3K4, H3K36 and H3K79, respectively). H3K4 di- and tri-methylation (H3K4me2/3) is

catalyzed by the Set1 complex (also called the COMPASS complex) and is associated with steady-

state gene transcription; thus, H3K4me2/3 is considered to be an ‘activating’ mark in mammals. Con-

versely, in budding yeast, most of the evidence indicates that H3K4 methylation is a repressive mark

(Shilatifard, 2006; Weiner et al., 2012). H3K36 tri-methylation (H3K36me3) by Set2 directs the

deacetylation of histones, predominantly at the 3’ portion of gene open reading frames (ORFs), to

suppress spurious intragenic transcription initiation (Carrozza et al., 2005). Methylation of H3K79

(H3K79me) affects telomeric heterochromatin structure because mutations at H3K79 as well as inac-

tivation of its methyltransferase, Dot1, lead to loss of telomere silencing (Jones et al., 2008;

Ng et al., 2002). The functions of each modification are largely dissected by using histone mutations

in combination with the inactivation of corresponding methyltransferases, under which circumstances

the modifications on both sister histones are simultaneously removed, making it difficult to study the

crosstalk between modifications on sister histones.

Although two copies of each histone in a nucleosome possess identical protein sequences, his-

tone-modification enzymes do not always modify sister histones simultaneously (van Rossum et al.,

2012; Voigt et al., 2013). For example, symmetrical modification of histone lysines within a

eLife digest Inside each human cell, about two meters of DNA is wrapped around millions of

proteins called histones, forming structures known as nucleosomes. Each nucleosome contains 147

letters of DNA code and two copies of four different histones – H2A, H2B, H3 and H4 – meaning

eight proteins in total.

The two copies of each histone protein found in a nucleosome are referred to as “sister” histones

and are identical. Histone proteins have long tails that the cell can edit by adding chemical groups

at specific positions. This changes the way the cell copies, uses and repairs its DNA. Previous studies

show that identical sister histones can end up with different modifications. But, it was not clear what

effect this had.

To adress this issue, there are two questions to answer. What do asymmetric sister histones do in

living cells? And, does a modification to one histone affect its sister? Gene editing could help

scientists to understand the effect of asymmetrical tail modification by forcing cells to make non-

identical sister histones. However, this is challenging because most animals studied in the laboratory

have many copies of the genes for histones. Fruit flies, for example, have 23 copies of their histone

genes. The single-celled yeast Saccharomyces cerevisiae has only two copies of its histone genes.

Yet, even if one of these genes was replaced with a mutant gene and the other left unedited or

“wild-type”, there would be nothing to stop the cell from forming nucleosomes in which both sister

histones were still identical – that is to say, mutant with mutant or wild-type with wild-type.

Now, Zhou, Liu et al. report a new method that allowed them to edit the tail sequence of one H3

histone but not its sister. First, they searched for, and found, a pair of mutant H3 genes, which

encode two extremely similar but different H3 proteins that could bind to each other but not to

themselves. As a result, yeast cells with the genes for these proteins could only form nucleosomes in

which the sister H3 histones were non-identical. Next, Zhou et al. made a small change to the tail of

one of the H3 sisters which meant it could not be modified. The resulting nucleosomes contain one

H3 histone with a wild-type tail and one with a mutant tail. The cell could only modify one of them,

mimicking natural asymmetrical modifications.

The new technique revealed that modification of one sister does not affect the the other. It also

revealed that modifications to sister histones can work both alone and together. In some cases, the

cell needs only edit one tail to affect the use of a gene. Other times, it must edit both tails for

greatest effect.

This new tool is the first step in understanding the contribution of the tails of sister histones in

living cells. In future, it should help to uncover the effect of different combinations of modifications.

This could shed light on how cells control the use of different genes.



Research article Genes and Chromosomes



nucleosome is not globally required in HeLa cells (Chen et al., 2011). In addition, in different cell

types, a significant number of nucleosomes contain asymmetrically modified sister histones

(Fisher and Fisher, 2011; Mikkelsen et al., 2007; Voigt et al., 2012). Furthermore, asymmetrically

modified nucleosomes are present in embryonic stem cells but are symmetrically modified upon dif-

ferentiation (Voigt et al., 2012). Each of these studies suggests that sister histones within a single

nucleosome may function independently in gene regulation. A synthetic system for the generation

of asymmetrically modified nucleosomes has been used to study histone PTM crosstalk in vitro

(Lechner et al., 2016), but the lack of a genetic model system for studying asymmetric histone mod-

ifications in vivo has prevented exploration of the biological significance of this previously docu-

mented phenomenon.

To investigate the individual contributions of sister histones and their modifications to chromatin

structure and function, we employed a protein engineering strategy to mutate both copies of his-

tone H3 in their interaction interface. After screening for mutants that were able to form histone H3

heterodimers but not H3 homodimers, we successfully set up a bivalent nucleosome system in the

budding yeast Saccharomyces cerevisiae. By using this unique system, we validated the establish-

ment of asymmetrically methylated H3K4, H3K36 or K3K79 in chromatin in yeast in vivo. Further-

more, we examined the functions of asymmetrically modified sister histones in the regulation of

chromatin structure and gene transcription. Our results revealed that modifications such as H3K4me,

H3K36me or K3K79me on sister histone H3s could be independent of each other. In addition, the

same modifications on both sister H3 histones can affect transcription in a cooperative, independent

or redundant manner. Our study provides the first picture of the individual contributions of sister his-

tones to chromatin dynamics in vivo.

Results

A bivalent nucleosome system to study sister histone H3s in yeastIn S. cerevisiae, each canonical histone is encoded by two genes. H3 is encoded by HHT1 and HHT2,

and H4 is encoded by HHF1 and HHF2. The histone genes are organized into a pair of divergently

transcribed loci with HHT1-HHF1 and HHT2-HHF2 linked together. Owing to redundancy, deletion

of either locus does not cause lethality (Dollard et al., 1994). As asymmetrical modifications

have previously been reported on histone H3 in vivo (Voigt et al., 2012), we began by examining

H3. Previous structural work revealed that two molecules of histone H3 interact through their car-

boxy-terminal four-helix bundle to form a homodimer (Luger et al., 1997; Ramachandran et al.,

2011; White et al., 2001) (Figure 1A). We performed site-directed mutagenesis on the Ala110,

Ala114 and Leu130 residues of the HHT1 gene. These residues were chosen because they were spa-

tially close and within the bundle region that interacts to form the H3 homodimer (Luger et al.,

1997; Ramachandran et al., 2011; White et al., 2001). These neutral amino acids were mutated to

acidic or basic residues to make them electronegative or electropositive under physiological condi-

tions. We reasoned that if we created an H3 allele with an electronegative (or electropositive) inter-

face, it would not form homodimers, but it would interact with a different H3 allele with an

electropositive (or electronegative) interface, thereby creating a heterodimer (Figure 1A).

Yeast cells lacking chromosomal HHT1 and HHT2 genes but containing the HHT1 gene on a

counter-selectable URA3 plasmid were transformed with plasmids carrying the mutated histone H3

genes. We then screened for histone H3 mutants that did not support cell viability when loss of the

wild-type (WT) HHT1 gene was counter-selected using 5-fluoroorotic acid (5-FOA) (Figure 1B). Only

the H3 mutant bearing the A110E mutation survived (Figure 1C), suggesting that the other 14 his-

tone H3 mutants could not form a homodimer. Next, yeast cells were co-transformed with the pair-

wise plasmids carrying these 14 mutated histone H3 genes (Figure 1D). Notably, only the H3A110D

and H3L130H pairing was able to support cell viability on a 5-FOA plate (Figure 1E; Figure 1—fig-

ure supplement 1), allowing us to infer that the H3A110D and H3L130H mutants form a hetero-

dimer that could be assembled into functional nucleosomes in vivo. Considering that histidine’s

positive charge is weakened under physiological pH conditions and may increase the risk for

H3L130H self-interaction, we used the relatively weaker ADE3 promoter to reduce the expression of

H3L130H (Agez et al., 2007; Antczak et al., 2006). This strain will be hereafter referred to as the

H3D/H3H strain.




YC -His 5-FOA

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Figure 1. The complementary mutants of histone H3A110D and H3L130H are assembled into nucleosomes in vivo.

(A) Schematic illustration of the production of histone H3 mutants that are able to complement and form

heterodimers. (B and D) Schematic strategy for screening histone H3 mutants. Genomic HHF1, HHF2 (encoding

histone H4), HHT1 and HHT2 (encoding histone H3) genes were knocked out, and yeast cells (LHT001 background)

were transformed with plasmids expressing wild-type (WT) or mutated histone H3 along with WT H4. (C) Histone

H3 mutants cannot survive without WT histone H3. Dotting assay was performed to examine the cell viability of

WT cells (LHT001) and 15 histone H3 mutants carrying pRS313-hht1a/b (HIS3 marker). (E) H3A110D and H3L130H

mutants can form a heterodimer. Mutants carrying both pRS313-hht1a (HIS3 marker) and pRS314-hht1b (TRP1

marker) were dotted on a 5-fluoroorotic acid (5-FOA) plate. WT and mutants that survived on 5-FOA plate are

highlighted in red.


The following figure supplement is available for figure 1:

Figure supplement 1. The pairwise histone H3 mutants other than H3A110D and H3L130H cannot grow when the

WT histone H3 gene is counter-selected using 5-FOA.






Characterization of the H3D/H3H strainTo confirm that mutant histones H3A110D and H3L130H equally assembled into nucleosomes, we

epitope-tagged one copy of H3 in H3D/H3H cells with Myc. After preparing mono-nucleosomes (Fig-

ure 2—figure supplement 1), we performed immunoprecipitations with an anti-Myc antibody and

examined both Myc-tagged and untagged histone H3. In the control, the chromatin from both the

myc-H3 strain and the untagged H3 strain was mixed, and the immunoprecipitation of mononucleo-

somes using the anti-Myc antibody did not pull down untagged H3 (Figure 2A, second lane). As the

anti-H3 N-terminal antibody could not recognize Myc-tagged histone H3 (Figure 2A), we normalized

immunoprecipitated myc-H3L130H and myc-H3A110D to the same level. The amounts of the co-

immunoprecipitated complementary H3A110D and H3L130H histones were identical (Figure 2A),

reflecting an equal incorporation of H3A110D and H3L130H into mononucleosomes in H3D/H3H

cells. Next, we examined the ratio of H3A110D to H3L130H and the nucleosome positioning at the

GAL1-10 gene locus in the H3D/H3H cells. GAL1-10 intergenic chromatin consists of a non-nucleoso-

mal, UAS-containing hypersensitive region (Lohr, 1984; Lohr and Hopper, 1985) surrounded by

positioned nucleosomes (Lohr and Lopez, 1995; Lohr et al., 1987). A chromatin immunoprecipita-

tion (ChIP) assay showed almost the same enrichment of H3A110D and H3L130H at the GAL1 gene

promoter (Figure 2B), supporting our conclusion that mutant histones H3A110D and H3L130H were

assembled into nucleosomes at a ratio of 1:1 in vivo. MNase digestion of the GAL1-10 promoter

revealed that the nucleosome array on the GAL10 side of the UAS region displayed a similar diges-

tion pattern in H3D/H3H and WT cells, but the nucleosome array on the GAL1 side showed a more

evenly digested pattern in WT cells than in H3D/H3H cells (Figure 2C), suggesting altered nucleo-

some stability in the GAL1 region in H3D/H3H cells.

We next determined the functional viability of the H3D/H3H mutant using the histone shuffle strain

(LHT001) as a WT control. H3D/H3H mutant and WT cells exhibited identical growth rates in yeast

extract peptone dextrose (YPD) medium at 23˚C, 30˚C and 37˚C. In addition, when H3D/H3H cells

were challenged by rapamycin (data not shown) or DNA-damage reagents, such as phleomycin or

methyl methanesulfonate (MMS), they showed nearly the same sensitivity as WT cells (Figure 2D).

Interestingly, compared with WT cells, H3D/H3H cells showed a reduced growth rate when cultured

in raffinose or glycerol medium (Figure 2E). We then checked the levels of multiple histone PTMs in

WT and H3D/H3H strains by western blot and found no significant differences (Figure 2F). Further,

we performed a genome-wide RNA-Seq assay to examine the gene expression profiles in WT and

H3D/H3H strains. Statistical analysis confirmed the reproducibility of the RNA-Seq results in each

strain (Figure 2—figure supplement 2). The global gene expression profile of the H3D/H3H strain

was found to be very similar to that of the WT strain (Figure 2G), but we did see some genes

with expression levels that varied between the H3D/H3Hand WT strains. Through Gene Ontology

analysis (see the Materials and methods for details), we found that most of the outliers were downre-

gulated by histone H3 mutations. Interestingly, the genes encoding cytochrome-c reductase activity

and ATPase activity were among the outliers (Tzagoloff et al., 1975) (Figure 2—source data 2).

This finding might provide an explanation for the reduced growth of the H3D/H3H strain when glyc-

erol was used as the carbon source (Figure 2E).

Taken together, the observations presented in Figure 2 indicated that the H3D/H3H strain

behaved similar to the WT strain under most, but not all of the tested circumstances; thus, this strain

provides a unique and valid system for analyzing asymmetrically modified sister histones.

N-terminal deletion of one sister histone H3 tail does not affect theother tailTo address whether there is crosstalk between the amino-terminal tails of sister histone H3s in one

nucleosome, we constructed strains that lacked the N-terminal 4–15 amino acids on one or both sis-

ter H3 histones (Mann and Grunstein, 1992). The H3DD4–15/H3H and H3D/H3HD4–15 strains con-

tained one copy of N-terminal-deleted H3, resulting in asymmetrically deleted histone H3

(Figure 3A). The H3DD4–15/H3HD4–15 strain containing two copies of N-terminal-deleted H3 was

also constructed and used as a negative control. The nucleosomes of the H3D/H3H (treated as WT

hereafter) and mutant strains were precipitated, and western blots were performed to examine the

levels of histone H3 N-terminal and K4 tri-methylation. Both histone H3 N-terminal and H3K4me3

signals in H3DD4–15/H3H and H3D/H3HD4–15 cells were reduced to approximately half of those




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Figure 2. Characterization of the H3D/H3H strain. (A) Mononucleosomes prepared from both the cells expressing Myc-tagged H3 (ZL38) and the cells

expressing untagged H3 (LHT001) were mixed (Myc-H3 +H3) and immunoprecipitated with the anti-Myc antibody. The same immunoprecipitation (IP)

assay was performed with mononucleosomes from strains bearing Myc-H3L130H/H3A110D (Myc-H3H/H3D) and Myc-H3A110D/H3L130H (Myc-H3D/H3H).

The precipitated mononucleosomes were denatured and examined by western blotting with both anti-Myc (Myc) and anti-histone H3 N-terminal

antibodies (H3N). (B) Chromatin IP (ChIP) analysis was performed in Myc-H3A110D/H3L130H (Myc-H3D/H3H) and Myc-H3L130H/H3A110D (Myc-H3H/

H3D) cells using anti-Myc antibody. The precipitated DNA was analyzed by qRT-PCR with primers specific for the GAL1-10 gene promoter and

normalized to the ACT1 gene. Error bars indicate s.e.m. for three independent experiments. (C) MNase digestion of nuclei from WT (LHT001) and H3D/

H3H strains. Nuclei were digested with increasing concentrations of MNase for 4 min. MNase cleavage sites were mapped from the EcoRI site within

Figure 2 continued on next page




Figure 2 continued

GAL10 by indirect end labeling analysis on a 1.6% agarose gel. Marker fragments are from PCR products of 1 kb and 500 bp in length. The UAS region

and nucleosome positions are schematically shown on the right. ND, naked DNA. (D) Dotting assays were performed in H3D/H3H mutant and WT

(LHT001) cells. Plates were photographed after incubation at 37˚C, 30˚C and 23˚C on yeast extract peptone dextrose (YPD) medium or after incubation

at 30˚C on YPD, YPD containing phleomycin and YPD containing MMS on Days 1, 2 and 3. (E) Growth curve assays were performed in H3D/H3H mutant

and WT cells for the indicated time in medium containing different carbon sources. (F) Yeast chromatins extracted from WT (LHT001) and H3D/H3H

strains were monitored by western blot analysis with antibodies against H3K4ac, H3K4me2, H3K4me3, H3K18ac, H3K27ac, H3K36me3, H3N (H3

N-terminal), H4ac, H4K16ac and H4. Signals are normalized by anti-H4 antibody. (G) Scatter plot showing the average Reads Per Kilobase per Million

mapped reads (RPKM) of two replicates distribution of the WT (LHT001) and H3D/H3H strains. The Pearson’s product-moment correlation of

Log10(WT_average_RPKM +1) and Log10(H3D/H3H _average_RPKM +1) is 0.9236. The red line is the fitted curve, which has a slope of 0.9966 and which

passes through the (0,0) point. R-square is 0.98 and p value�2.2e-16 (see Materials and methods for details).


The following source data and figure supplements are available for figure 2:

Source data 1. Characterization of the H3D/H3H strain.


Source data 2. Analysis of RNA-Seq data.


Figure supplement 1. Mononucleosome preparation from H3D/H3H cells.


Figure supplement 2. Sample-to-sample reproducibility for the RNA-Seq assay of WT (LHT001) and H3D/H3H strains.


A

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Figure 3. Examination of an asymmetric deletion in the N-terminus of histone H3. (A) Schematic illustration of

asymmetrical N-terminal-deleted histone H3 mutants of H3DD4–15/H3H, H3D/H3HD4–15 and H3DD4–15/H3HD4–15

derived from the H3D/H3H strain. The genotype of each mutant is labeled in a different color, and these colors are

applied to identify each mutant in all the panels in this figure. (B) Asymmetric H3 N-terminal-deletion is

successfully established on chromatin. Nucleosomes were immunoprecipitated by the anti-H2B antibody from cells

of the yeast strains in (A) and analyzed by western blotting using the anti-H3 N-terminal antibody, the anti-

H3K4me3 antibody and, as a normalization, the anti-H4 antibody. (C) Quantification of the H3N and H3K4me3

signals in (B) as mean ratio relative to H3D/H3H and normalized to H4 signals. (See the Materials and methods for

quantification details.)


The following source data is available for figure 3:

Source data 1. Examination of an asymmetric deletion in the N-terminus of histone H3.









observed in H3D/H3H cells (Figure 3B and C). These results indicated that H3 N-terminal deletion on

one sister H3 did not influence H3K4 methylation on the other.

K4 me2/3 on sister H3s independently regulates the transcriptionefficiency of GAL1 upon inductionAs the genes for H3A110D and H3L130H encoded compatible and functional histone H3 proteins,

we anticipated that the substitution of K with R on one sister H3 would largely mimic unmethylated

K. Thus, asymmetrically modified nucleosomes could be assembled in chromatin in vivo. To test this

idea, we first introduced the K4R mutation into H3A110D (H3DK4R) or H3L130H (H3HK4R) in the

H3D/H3H strain (Figure 4A). Western blotting showed that H3K4me3 in H3DK4R/H3H or H3D/

H3HK4R cells was approximately 50% lower than that in H3D/H3H cells, whereas little difference in

H3K36me3 was detected among the tested strains (Figure 4B and C). Therefore, these results sug-

gest that the hybrid strains contain only mimics of asymmetrically deposited K4me3. For sister H3

histones in a nucleosome, a lack of K4me3 in one tail did not influence K4me3 in the other tail,

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Figure 4. Asymmetrically methylated K4 on sister H3s independently upregulates GAL1 transcription efficiency. (A) Schematic illustration of

asymmetrical mutation of histone H3K4R on either H3A110D (H3D) or H3L130H (H3H). The K to R mutation is marked by Ò on the histone H3 tails. The

color labeling of each strain is applied for all the corresponding panels. (B) Asymmetrical K4me3 marks on sister H3s are successfully established on

chromatin. Nucleosomes were immunoprecipitated by the anti-H2B antibody from isogenic strains and analyzed by western blotting using anti-

H3K4me3, anti-H3K36me3 and, as a normalization, anti-H4 antibodies. (C) Quantification of western blotting signals for H3K4me3 as mean ratio relative

to H3D/H3H and normalized to signals for H4. (D) The cells with asymmetrical sister K4me3 show an intermediate level of GAL1 gene expression. Yeast

strains illustrated in (A) were subject to galactose induction. Total RNA was prepared at the indicated time points and analyzed by real-time

quantitative PCR (qRT-PCR) with primers specific for GAL1 and normalized by ACT1. (E) An alternative representation of the data in (D) at 30 min is

expressed as mean ratio to H3D/H3H, whose level was set to 1. (F) Asymmetric K4me3 on sister H3s is established in GAL1. ChIP experiments were

performed on the promoter of GAL1 in the indicated yeast strains using the anti-H3K4me3 antibody, and values are normalized to histone H4. (G) The

levels of Gal4 recruitment are inversely proportional to the levels of K4me3. Gal4 was tagged with 13 � myc, and ChIP experiments were performed on

the UAS region of GAL1 in the indicated yeast strains with anti-Myc antibody. (H) Detection of GAL1 levels when SPP1, SDC1 or SET1 is deleted. SPP1,

SDC1 or SET1, respectively, was knocked out in the indicated strains, which were subject to galactose induction. RNA was extracted, analyzed and

expressed as in (E). H3D/H3H cells are regarded as WT controls. All of the ChIP values are expressed as mean ratio to H3D/H3H, whose level was set to

1. All error bars indicate s.e.m. for at least duplicated experiments.



Source data 1. Asymmetrically methylated K4 on sister H3s independently upregulates GAL1 transcription efficiency.







consistent with the observation in Figure 3B. In addition, H3K4me3 and H3K36me3 were indepen-

dent of each other because the decrease in H3K4me3 did not alter the level of H3K36me3.

Cells that lack histone H3K4 methylation have an increased GAL1 induction level (Pinskaya et al.,

2009). To assess the effect of asymmetrical H3K4me3 on transcription, we assessed GAL1 mRNA

levels in K4R mutant cells. Compared with H3D/H3H cells, H3DK4R/H3H and H3D/H3HK4R single-tail

mutant cells showed a two-fold increase in GAL1 mRNA levels. Compared with single-tail mutant

cells, H3DK4R/H3HK4R double-tail mutant cells showed a further two-fold increase in GAL1 mRNA

levels (Figure 4D and E). GAL1 mRNA levels were inversely proportional to H3K4me3 levels at the

GAL1 promoter (Figure 4F), suggesting a tight correlation between induction levels and H3K4me3

abundance. We also examined the enrichment of Gal4 binding to the GAL1 promoter using a ChIP

assay. Gal4 is the primary activator of GAL1 transcription (Johnston, 1987). A moderate level of

Gal4 recruitment to the GAL1 promoter was observed in the asymmetrical H3DK4R/H3H and H3D/

H3HK4R mutant strains compared with that in their symmetrical H3D/H3H and H3DK4R/H3HK4R coun-

terparts (Figure 4G). Therefore, each K4me3-modified sister histone H3 contributed

independently to GAL1 gene transcription, which is probably recognized and read by the GAL1 acti-

vator Gal4.

Set1C in yeast contains eight subunits, including Set1, Spp1 and Sdc1, and is responsible for

methylating histone H3K4 (Dehe and Geli, 2006; Roguev et al., 2001). Deletion of SET1 eliminates

H3K4 mono-, di- and tri-methylation; deletion of SPP1 affects only H3K4 tri-methylation; and dele-

tion of SDC1 affects di- and tri-methylation of H3K4 (Pinskaya et al., 2009). To address which type

of asymmetrical H3K4 methylation affects GAL1 transcription, and to confirm that the changes in

gene expression were due to asymmetrical H3K4 methylation instead of the K4R mutation, we

knocked out SET1, SPP1 and SDC1 in the H3D/H3H, H3DK4R/H3H, H3D/H3HK4R and H3DK4R/

H3HK4R strains and examined GAL1 levels in galactose medium. As the data show, loss of SPP1,

SDC1 and SET1 led to the upregulation of GAL1 transcription, which is consistent with previous find-

ings (Pinskaya et al., 2009). Meanwhile, an intermediate level of GAL1 expression was seen in

spp1D H3DK4R/H3H and spp1D H3D/H3HK4R cells, whereas no significant difference was found in

either sdc1D or set1D mutants (Figure 4H). As distinguishing between the effects of H3K4me2 and

H3K4me3 is difficult, we concluded that H3K4me2/3 but not mono-methylation of H3K4 on sister

H3s contributed the most to GAL1 regulation.

K36 methylation on sister H3s independently regulates transcriptioninitiation fidelityAs both asymmetrical H3 N-terminal deletion and asymmetrical H3K4me3 were successfully assem-

bled in chromatin, we constructed mutants that mimicked asymmetrical H3K36me. A K36R mutation

was introduced into H3A110D (H3DK36R) or H3L130H (H3HK36R) in the H3D/H3H strain (Figure 5A).

The level of H3K36me3 and H3K4me3 on chromatin was examined by western blotting.

When compared with H3D/H3H cells, H3DK36R/H3H or H3D/H3HK36R cells showed an approximately

50% decrease in H3K36me3, whereas little difference in H3K4me3 was detected among the tested

strains (Figure 5B and C). These data indicated that the H3DK36R/H3H or H3D/H3HK36R mutants

contained only mimics of asymmetrically deposited K36me3, and loss of K36me3 on one tail did not

affect K36me3 on the other tail. In addition, in agreement with the data shown in Figure 4B,

H3K36me3 and H3K4me were independently regulated chromatin modifications.

H3K36me3 directs deacetylation of histone H4 in gene-coding regions to suppress spurious intra-

genic transcription (Carrozza et al., 2005). To address whether H3K36me3 on both sister histone

H3s contributed to the regulation of cryptic transcription, we tested the level of intragenic initiation

in the H3K36R mutants within the FLO8, PCA1 and STE11 genes. Each of these genes is regulated

by K36 methylation. Northern blot analysis showed that the loss of K36 methylation on H3 tails

resulted in short transcripts of the tested genes, consistent with previous findings (Carrozza et al.,

2005; Li et al., 2007). Compared with H3D/H3H cells and symmetrically mutated H3K36 cells, asym-

metrical H3DK36R/H3H and H3D/H3HK36R cells exhibited an intermediate level of short transcripts

(Figure 5D). We next used anti-acetylated histone H4 antibodies to perform a ChIP assay on the 3’

ORF of the FLO8, PCA1 and STE11 genes. H4 acetylation (H4ac) levels in H3DK36R/H3H and H3D/

H3HK36R cells were intermediate relative to those in H3D/H3H cells and symmetrically mutated

H3K36 cells. Moreover, H4ac levels were inversely correlated with H3K36me3 levels in the same

region (Figure 5E and F). In the absence of Set2, the level of H4ac in the tested genes showed no




0.0

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Figure 5. The H3K36me3 marks on sister histones regulate transcription independently. (A) Schematic illustration

of asymmetrical mutation of histone H3K36R on either H3A110D (H3D) or H3L130H (H3H). The K to R mutation is

marked by Ò on the histone H3. The color labeling of each strain is applied for all the corresponding panels. (B)

Asymmetrical K36me3 on sister H3s is successfully established on chromatin. Nucleosomes were





significant differences in H3D/H3H, H3DK36R/H3H, H3D/H3HK36R and H3DK36R/H3HK36R cells

(Figure 5G). These observations indicated that the regulation of accurate transcription initiation was

sensitive to the magnitude of H3K36me3. Accordingly, H4ac levels were regulated by H3K36me3 on

both sister histones. In light of these data, we concluded that H3K36me3 on either sister histone

played an independent regulatory role in suppressing spurious intragenic transcription.

K79 methylation on sister H3s cooperatively regulates gene silencing intelomeric regionsH3K79 methylation regulates gene silencing in some telomere-proximal regions (Takahashi et al.,

2011). To address whether the H3K79 methylation of both sister histones is required to maintain

silent chromatin near telomeres, we used strains in which H3K79 could be methylated at either one

(H3DK79R/H3H and H3D/H3HK79R) or none (H3DK79R/H3HK79R) of the H3 sister histones

(Figure 6A). Western blot analysis revealed that H3K79me2/3 levels in H3DK79R/H3H and H3D/

H3HK79R cells were approximately 50% lower than those in H3D/H3H cells (Figure 6B and C), sug-

gesting the incorporation of asymmetrical H3K79me into chromatin and that the methylation of K79

occurs independently on each sister H3.

We examined the transcription levels of COS12, ERR1 and ERR3, which are located proximal to

the telomeric ends of chromosomes VIIL, XVR and XIIIR, respectively (Takahashi et al., 2011). As

expected, the K79R mutations on both sister H3s resulted in decreased silencing of those genes.

Surprisingly, H3DK79R/H3H and H3D/H3HK79R cells containing asymmetrical H3K79me exhibited the

same level of silencing loss as that of H3DK79R/H3HK79R or sir2D cells (Figure 6D). A ChIP experi-

ment confirmed that K79me levels at the promoters of the genes tested in H3DK79R/H3H and H3D/

H3HK79R cells decreased to approximately half of those in H3D/H3H cells (Figure 6E). Accordingly,

the H4ac level in the ORF region was upregulated in K79R mutated cells (Figure 6F). Collectively,

these data reveal that K79me marks on both sister H3s act cooperatively to maintain gene silencing

near telomeres.

Cell sensitivity to genotoxic agents is affected by sister histone H3K4,H3K36 and H3K79 modificationsH3K4, H3K36 and H3K79 methylation affects DNA double-strand break (DSB) repair (Faucher and

Wellinger, 2010; Jha and Strahl, 2014; Pai et al., 2014). Therefore, we examined the regulatory

role of asymmetric H3K4, H3K36 or H3K79 methylation in DSB repair. Mutant cells bearing asymmet-

rical methylated or non-methylated H3K4, H3K36 or H3K79 were serially diluted and spotted onto

plates containing various genotoxic chemicals, including phleomycin, hydroxyurea (HU) or MMS.

H3K4R or H3K79R mutations on either one or two sister histones reduced cell growth in the

Figure 5 continued

immunoprecipitated by anti-H2B antibody from isogenic strains, as identified by colored numbers, and analyzed

by western blot using anti-H3K4me3, anti-H3K36me3 and, as a normalization, anti-H4 antibody. (C) Quantification

of western blot signals for H3K36me3 as mean ratio relative to H3D/H3H and normalized to signals for H4. (D)

Northern blot analysis of the FLO8, STE11 and PCA1 transcripts in H3K36R mutants. RNA from H3D/H3H,

H3DK36R/H3H, H3D/H3HK36R and H3DK36R/H3HK36R strains was probed with sequences complementary to the 3’

region of FLO8, STE11, PCA1 and, as a loading control, SCR1. The full-length (FL) and short transcript signals are

indicated. (E and F) Asymmetric K36me3 on sister H3s results in an intermediate level of H4ac in FLO8, STE11 and

PCA1. ChIP experiments were performed in 3’ ORF region of FLO8, STE11 and PCA1 in the indicated cells with

anti-H3K36me3 antibody (E) and anti-H4ac antibody (F). Values are normalized to histone H4 and expressed as

mean ratio to H3D/H3H. (G) H4ac level of the FLO8, STE11 and PCA1 loci in set2D cells bearing different K36me3

states on sister H3s. SET2 was knocked out in the indicated cells and ChIP experiments were performed as in (E).

Values are normalized to histone H4 and expressed as mean ratio to H3D/H3H. The H3D/H3H cells are regarded as

a WT control. In all cases, the values of H3D/H3Hare set to 1. All error bars indicate s.e.m. for at least duplicated

experiments.



Source data 1. The H3K36me3 marks on sister histones regulate transcription independently.







H3DK79R/H3H

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Figure 6. The H3K79me marks on both sister histones contribute to the regulation of sub-telomeric chromatin

structure. (A) Schematic illustration of the asymmetrical mutation of histone H3K79R on either H3A110D (H3D) or

H3L130H (H3H). The K to R mutation is marked by on the histone H3. The color labeling of each strain is applied in

the other panels in this figure. (B) Asymmetrical K79me2/3 on sister H3s is established on chromatin. Nucleosomes

were immunoprecipitated by anti-H2B antibody from isogenic strains (identified by colored numbers) and analyzed

by western blot using anti-H3K79me2/3, anti-H3K4me3 and, as a normalization, anti-H4 antibody. (C)

Quantification of western blot signals for H3K79me2/3 as mean ratio relative to H3D/H3H. (D) Both H3K79me marks

on sister H3s are required for the maintenance of telomere silencing. Total RNA was prepared and analyzed by

real-time quantitative PCR (qRT-PCR) with primers specific for COS12, ERR1 and ERR3 and normalized by ACT1.

The black bar represents data for samples of sir2D H3D/H3H mutants, acting as a positive control. Values are





presence of the tested genotoxins. Notably, the H3DK36R/H3HK36R mutant was hypersensitive to

phleomycin and mildly sensitive to MMS. Compared with the wild type (H3D/H3H) and correspond-

ing double-tail mutant, single-tail H3K36R or H3K79R mutants displayed an intermediate level of

sensitivity to the genotoxic agents. The H3K4 mutants showed a similar level of sensitivity to HU and

MMS, but single-tail H3K4R mutants displayed less growth in response to phleomycin treatment

than did double-tail H3K4R mutants (Figure 7).

Figure 6 continued

calculated and expressed as in Figure 4E. (E and F) Detection of K79me2/3 (E) and H4ac (F) at the promoters of

the COS12, ERR1 and ERR3. ChIP experiments were performed on the promoters of COS12, ERR1 and ERR3 in the

indicated cells with anti-H3K79me2/3 antibody (E) and anti-H4ac antibody (F). Values are normalized to histone H4

and expressed as mean ratio to H3D/H3H. All error bars indicate s.e.m. for at least duplicated experiments.



Source data 1. The H3K79me marks on both sister histones contribute to the regulation of sub-telomeric chroma-

tin structure.


H3D/H3H

H3DK4R/H3H

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YPD, 30

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Phleomycin 5µg/ml

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HU 25mM

Day 3

H3D/H3H

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H3D/H3HK4R

H3DK4R/H3HK4R

H3DK36R/H3H

H3D/H3HK36R

H3DK79R/H3H

H3D/H3HK79R

H3DK79R/H3HK79R

H3DK36R/H3HK36R

0.01% MMS

Day 1

0.01% MMS

Day 2

0.01% MMS

Day 3

Figure 7. The performance of histone mutants challenged by multiple DNA damage reagents. Dotting assay was performed in the indicated mutants

as in Figure 2D. Plates were photographed after incubation at 30˚C on YPD medium or YPD medium containing DNA damage reagents, that is, 5 mg/

ml phleomycin, 25 mM HU and 0.01% MMS, for 1, 2 and 3 day(s).








Together, these observations suggest that, in response to DNA damage, H3K36me and

H3K79me marks on sister histones functioned independently, whereas H3K4me marks on sister his-

tones functioned cooperatively. Because the type of DNA damage triggered by the different geno-

toxic agents and the mechanisms of repair differ, we propose that the combination of sister histone

modifications may influence DNA repair in different ways.

Genome-wide analysis of gene expression in mutants withasymmetrically methylated sister H3K4 under glucose starvationChromatin regulators do not appear to affect steady-state transcription, but instead are required for

transcriptional reprogramming induced by environmental cues (Weiner et al., 2012). For example,

the genome-wide gene transcription profile of H3K4A cells was nearly the same as that of WT cells

when the cells were grown under normal conditions, whereas differences were observed when the

cells were challenged by multiple stress conditions (Weiner et al., 2012). To further unravel the

genome-wide function of sister H3K4me on transcription, we shifted the cultures of H3K4R mutants

and H3D/H3H strains from 2% to 0.05% glucose in the medium, which mimics calorie restriction.

After the cells were grown in 0.05% glucose medium for an hour, we performed RNA-Seq to exam-

ine the genome-wide gene-induction profiles, which are presented as fold-change (level of induc-

tion). The fold-change value refers to the level of transcription in the induced strains divided by that

in the uninduced strains. Of the 6000 genes in the yeast genome, approximately 2500 were altered

by the H3K4 to R mutation in response to glucose starvation. Over 1500 genes’ fold-change (MID,

as defined in Materials and methods) in both asymmetrical K4R mutants (H3DK4R/H3H and H3D/

H3HK4R) fell between those of H3D/H3H cells and double K4R mutants (Figure 8—figure supple-

ment 1A).

Statistical analysis by t-test and a gene skewness score (GSS) model described in the Materials

and methods revealed that 22 genes’ fold-changes in asymmetrical K4R mutant (H3DK4R/H3H and

H3D/H3HK4R) cells were nearly the same as those in symmetrical K4R mutant (H3DK4R/H3HK4R) cells

(Figure 8A,B; Figure 8—figure supplement 1B, Cluster I), indicating cooperativity of sister K4me at

these loci. The fold-changes of 191 genes in asymmetrical K4R mutant (H3DK4R/H3H and H3D/

H3HK4R) cells exhibited an intermediate state between those in symmetrical K4R mutant (H3DK4R/

H3HK4R) and WT(H3D/H3H) cells (p<0.05) (Figure 8A,B; Figure 8—figure supplement 1B, Cluster

II), suggesting that K4me on sister H3s independently regulates the expression of these genes. The

fold-changes of 158 genes in asymmetrical K4R mutant (H3DK4R/H3H and H3D/H3HK4R) cells were

nearly the same as those in WT H3D/H3H cells (Figure 8A,B; Figure 8—figure supplement 1B, Clus-

ter III), indicating redundancy of sister K4me at these loci. An approximately 50% decrease in

H3K4me3 in asymmetrical K4R mutants was confirmed in the 5’ ORFs of the YOR008C, YMR315W

and YLR359W genes, which belong to the three clusters (Figure 8—figure supplement 1C–E).

These assessments suggest that, in response to glucose starvation stress, H3K4me on two sister his-

tones in different gene loci impose their effects on transcription in a cooperative (e.g., Cluster I),

independent (e.g., Cluster II) or redundant (e.g., Cluster III) manner. Interestingly, the genes in Clus-

ter I and II were mostly upregulated (log2fold change >0), while the genes in Cluster III were mostly

downregulated (log2fold change <0) (Figure 8B), suggesting that under glucose starvation stress,

the transcription of upregulated genes may require more subtle regulation mechanisms, such as

asymmetrical modification of sister histones.

Although the H3K4R mutation in chromatin largely mimics K4me0, it is not the same as K4me0,

and the phenotypes seen in H3DK4R/H3H and H3D/H3HK4R cells might not result from loss of K4me.

To address this issue, we examined the genome-wide gene expression profile of set1D cells under

glucose starvation, and compared its fold-change with that of K4R mutants (Figure 8—figure sup-

plement 1F). Many of the genes in Clusters I, II and III overlapped with genes that are regulated by

SET1 deletion (Figure 8C, IT

set1D, IIT

set1D and IIIT

set1D, respectively), indicating that these over-

lapping genes are most probably regulated by K4me rather than the K4R mutation on sister H3s.

Genes modified by sister H3K4me under glucose starvation areclustered in pathways associated with glycometabolismTo determine which pathways were regulated by asymmetrical K4me on sister H3s in response to

glucose starvation, we carried out KEGG pathway analysis (Huang et al., 2009a, 2009b). Nine of




-4

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Skewness to H3D/H3H or H3DK4R/H3HK4R

H3DK4R/H3H

H3DK4R/H3HK4R H3D/H3H

I II III

I

13

9

set1∆

1447

II

137

54 45III

113

I ∩ set1∆II ∩ set1∆

III ∩ set1∆

Biosynthesis of

antibiotics (5)

TCA cycle (3)

Fructose and mannose

Metabolism (3)

Carbon metabolism (6)

Biosynthesis of

secondary

metabolites (6)

II ∩ set1∆

D

H3DK4R/H3HK4R ∩ set1∆

Biosynthesis of

secondary

metabolites (62)

Metabolic pathways (114)

Biosynthesis of

antibiotics (50)

Carbon metabolism (30)

Biosynthesis of

amino acids(25)

Oxidative

phosphorylation (14)

Pyruvate metabolism (13)

TCA cycle (12)

Others (110)

E

Figure 8. Genes in glycometabolism pathways are regulated by asymmetrically methylated K4 on sister H3s in

response to glucose starvation. (A) Histogram showing the gene skewness score (GSS) of asymmetrical K4R

mutants in set MID calculated by a GSS model (see the Materials and methods for details). Bidirectional arrows

with gradient colors indicate the increasing skewness of log2 fold change in asymmetrical K4R mutants to those in

either H3D/H3H (red end) or H3DK4R/H3HK4R (blue end) cells. Genes are classified to three clusters (I, II and III) by

t-test according to the protocols described in the Materials and methods. (B) Boxplots showing the gene’s fold-

changes of H3D/H3H and K4R mutants in three gene clusters (blue, cluster I; purple, cluster II; red, cluster III). The

log2 fold-change values are calculated as described in the Materials and methods. (C) Venn diagram showing the

overlaps between genes whose fold-change values are significantly altered by SET1 knockout in the H3D/H3H

strain in response to glucose starvation (green circle), and the genes in clusters I (blue circle), II (purple circle), and

III (red circle), respectively. (D and E) Pathways regulated by asymmetrically methylated K4 on sister H3s in

response to glucose starvation. Pie charts show the pathways with which the genes are specifically associated,

under the regulation of asymmetrical K4me on sister H3s (D), or under the regulation of K4me (E).


The following source data and figure supplement are available for figure 8:

Source data 1. Genes in glycometabolism pathways are regulated by asymmetrically methylated K4 on sister H3s

in response to glucose starvation.






the genes in the IT

set1D group and 45 of the genes in the IIIT

set1D group could not be mapped to

any specific pathways in the KEGG database. Remarkably, 54 of the genes in the IIT

set1D group,

which is regulated by sister H3K4me, were enriched in the pathways involved in glycometabolism,

such as carbon metabolism, TCA cycle, and fructose and mannose metabolism (Figure 8D). KEGG

pathway analysis was performed on genes that were regulated by both the H3DK4R/H3HK4R muta-

tion and SET1 deletion under glucose starvation. Interestingly, the three pathways involved in glyco-

metabolism in the IIT

set1D were also found in the list (Figure 8E). Therefore, under glucose

starvation stress, a significant proportion of H3K4me-responsive genes are regulated by the fluctua-

tion of H3K4me levels on sister H3s. Further analysis of the fold-changes of the genes in the glyco-

metabolism-associated pathways revealed a pattern similar to that of Cluster II (Figure 8—figure

supplement 1G), suggesting that the independent regulatory mode of sister H3K4me is an impor-

tant player in response to glucose starvation stress. Collectively, these results support the notions

that the on-off regulatory mode for H3K4me is more likely to be applicable to the transcription of

genes that do not specifically respond to external stimuli (e.g., genes in the IT

set1D and IIIT

set1D

groups), whereas the fine-tuning mode evolved to regulate the transcription of genes involved in

stress-responsive pathways (e.g., genes in the IIT

set1D group).

DiscussionIn a nucleosome, two canonical sister histones display identical sequences, suggesting that they

have evolved to play similar roles in the regulation of chromatin structure and function. However,

similar to post-translational chemical modifications on any protein, the modifications of histones pro-

vide an additional layer of chromatin regulation. Paradoxically, sister histones with either asymmetri-

cal modification or coexistence of activation and repression marks have been found in different cell

types (Fisher and Fisher, 2011; Mikkelsen et al., 2007; Voigt et al., 2012), raising the possibility

that sister histones in a single nucleosome may function independently. In this study, we took advan-

tage of a yeast system that allows for facile genetic manipulation of histones. We identified H3 muta-

tions that prevented homodimer formation and allowed heterodimer formation. After a series of

intentional and systematic screenings, we established a bivalent nucleosome system that enabled us

to express and monitor sister histone H3s independently in vivo. Owing to the nature of the nucleo-

some, which is the basic unit structure of chromatin, any possible indirect effect(s) caused by knock-

ing-in a histone mutation cannot be fully excluded from our analysis. Indeed compared with the

parental strain, the H3D/H3H strain did display some minor differences in carbon source preference

and nucleosome positioning (e.g., the GAL1 locus) (Figure 2C,E). In addition, when a mutation was

introduced into one of the sister histone H3s, only half of the H3 could be modified. Therefore, the

global level of modified H3 had a maximum value of 50% of the maximum in a normal cell. Discrimi-

nating between the biological consequences caused by forced asymmetrical modification and a

forced decrease in total modification is difficult. Nevertheless, we have provided biochemical and

functional evidence indicating that this unique genetic system is useful for studying asymmetrical

modifications on sister histones.

Figure 8 continued


Source data 2. Genes regulated by H3K4R mutation under glucose starvation.


Source data 3. GSS of K4R mutants.


Source data 4. Gene type classification.


Source data 5. KEGG pathway analysis of RNA-Seq data.


Figure supplement 1. Genes in glycometabolism pathways are regulated by asymmetrically methylated K4 on

sister H3s in response to glucose starvation.






Using this system, we found that histone H3K4 methylation on one tail is independent of the

other tail on the sister H3 histone (Figure 3B), suggesting that Set1C binds and modifies one tail in

a cis fashion. Consistently, mutation of K4R in one of the sister H3s did not affect the methylation of

K4 on the other tail (Figure 4B). Interestingly, the methylation of K36 or K79 on two sister H3s was

also independent (Figures 5B and 6B). The results of our genetic models are consistent with a previ-

ous observation that sister histones are not always modified in the same manner simultaneously

(Chen et al., 2011).

Asymmetrically modified nucleosomes exist on chromatin (Fisher and Fisher, 2011;

Mikkelsen et al., 2007; Voigt et al., 2012), but whether these asymmetrical modifications on sister

histones function in manner similar to or different from that of symmetrical modifications remains

largely unknown. In our study, we observed that K79me on both sister H3 histones was required for

silencing telomere-proximal genes through regulation of the acetylation level of histone H4

(Figure 6D), establishing a cooperative role for both sister histones in vivo. This mode of regulation

was also seen for the genes in the IT

set1D group when the cells were challenged with glucose star-

vation (Figure 8C). Different from K79me, H3K36me3 on two sister histone H3s did not appear to

have a synergistic effect but rather had an additive effect on suppressing spurious transcription

(Figure 5D), indicating that two K36me3 marks on sister histone H3s altered chromatin structure

independently. The same additive effect was observed in the genes grouped in IIT

set1D

(Figure 8C), as well as in GAL1 transcription levels (Figure 4E). In addition, K4me marks on sister his-

tone H3s redundantly affected the transcription of the genes grouped in IIIT

set1D (Figure 8C). Con-

sistent with our observations in transcription, sister H3K4me exhibited different regulatory modes in

response to various DNA-damage reagents (Figure 7). Thus, our data indicate that modifications on

sister histones could employ a cooperative, independent, or redundant mode of regulation of chro-

matin-associated processes. However, why the genes in different loci are subjected to different regu-

latory mechanisms remains unclear. One possibility is that different gene loci are targeted by

different readers, such as activators and repressors that sense the magnitude of H3K4me differently

during transcription. This hypothesis is supported by the data in Figure 4F and G showing that dif-

ferential marks of K4me3 on two sister histone H3s affected the enrichment of Gal4-activator binding

to the GAL1 gene promoter, thereby fine-tuning the transcription of GAL1. The chromatin readers

for different genomic loci have not yet been well characterized, so providing a mechanistic explana-

tion for the different performances of sister histone modifications in every case is difficult.

Histones and their modifications are unique to eukaryotes, and they are important in the packag-

ing of DNA into chromatin. From an evolutionary point of view, it may be that the possesion of two

identical copies of each histone in the chromatin in eukaryotes rather than one copy is a sporadic

outcome of natural selection. Previous high-throughput analysis showed that epigenetic regulation

in the form of histone modification plays a far more pronounced role during gene induction/repres-

sion than during steady-state expression (Weiner et al., 2012), suggesting the involvement of his-

tone modifications in regulation of gene expression in response to changing environmental cues. In

this study, we imposed glucose starvation on yeast cells to mimic an environmental cue. In response,

the additive effect of H3K4me on gene transcription was recapitulated in the groups of genes

that are enriched in pathways related to glycometabolism, such as carbon metabolism, TCA cycle,

and fructose and mannose metabolism (Figure 8D). These observations support the notion that in

order to adapt to environmental stress, sister histones execute their fine-tuning regulation by differ-

ential modifications.

In conclusion, this study provides new insights into how sister histones regulate the plasticity of

chromatin structure, as well as gene transcription, and how epigenetic regulation evolves to address

variable environmental cues. Given that combinatorial manipulations of sister histone H3 tails have

encountered technical challenges in other model systems, the bivalent nucleosome system that we

created in this study will be instrumental in further uncovering the role that combinatorial histone H3

modification crosstalk plays in regulating gene expression. In addition, our system for the genetic

manipulation of sister histone H3s could be extended to an asymmetry study of sister histone H4s,

which have N-terminal tail acetylations representing important epigenetic marks in various biological

processes. Moreover, the genetic system that we created will be useful in examining the role

that sister histones play in other biological processes, such as DNA repair and recombination, chro-

matin replication and heterochromatin assembly. Finally, since the protein sequences of histone H3s

are highly conserved during evolution, it will be appealing to apply the same scheme to construct a




bivalent nucleosome system in other model systems. However, the challenge might be much greater

in higher eukaryotes because the copy numbers of histone genes in these organisms are much

higher than those in yeast.

Materials and methods

Strains, antibodies and growth conditionsAll yeast strains used in this study were derived from yeast strain YPH500 (Sikorski and Hieter,

1989). The genotypes of the yeast strains are listed in Supplementary file 1. The native promoter of

HHT1 (L130H) in the strains derived from H3D/H3H was replaced with the ADE3 promoter. The his-

tone shuffle strain (LHT001) was constructed previously in our lab. Antibodies used in this study are

listed in Supplementary file 2.

For galactose induction assays, cells were grown in YPD (10 g/L yeast extract, 20 g/L peptone,

2% dextrose) to mid-log phase (OD600 = 0.4–0.6) before being shifted to medium containing raffi-

nose (10 g/L yeast extract, 20 g/L peptone, 2% raffinose) overnight. Each sample was induced by 2%

galactose for 10–30 min. Remaining samples in raffinose medium were taken as having an induction

time of 0 min.

For glucose starvation assays, samples were grown in YPD (2% glucose) to mid-log phase and

then shifted to medium containing 0.05% glucose for one hour.

Mononucleosome preparation and immunoprecipitationYeast cells were cross-linked with 1% formaldehyde for 15 min at room temperature and then resus-

pended in lysis buffer (50 mM HEPES [pH 7.5], 35 mM NaCl, 0.5% Na-Deoxycholate [wt/vol], 5 mM

EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride [PMSF], protease inhibitor cocktail).

Cells were lysed using glass beads and sonicated to shear the chromatin to fragment sizes of 200–

400 bp. After centrifugation at 10,000 g for 10 min, the supernatant fraction was subjected to further

fractionation with a 24 ml Superdex-200 column (GE) in IP buffer (10 mM Tris-HCl [pH 8.0], 100 mM

NaCl, 0.5 mM EDTA, 1 mM DTT). Fractions containing mononucleosomes were pooled for subse-

quent incubation with anti-Myc antibody and protein G sepharose beads (GE) overnight at 4˚C. Thebeads were washed with wash buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 0.5% Na-Deoxycholate

[wt/vol], 5 mM EDTA, 1% Triton X-100) and TE (10 mM Tris-HCl [pH 8.0], 1 mM EDTA). Finally, the

immunoprecipitated mononucleosomes were eluted from beads with elution buffer (10 mM Tris-HCl

[pH 8.0], 1 mM EDTA, 1% SDS [wt/vol]).

Quantitative reverse transcription-PCR (qRT-PCR)Total RNA was isolated from yeast cells with an RNeasy mini kit (Qiagen). cDNA was synthesized

using the Fastquant RT kit (Tiangen). 1 ml of the RT reaction was used in the subsequent real-time

fluorescence quantitative PCR (ABI). Primer pairs used in qRT-PCR were listed in Supplementary file

3.

The expression of GAL1 was normalized to the RNA levels of ACT1, and the fold-changes were

calculated by defining the relative mRNA level at 0 min as 1.

MNase digestion assay and Southern blottingPreparation and digestion of yeast nuclei were performed as described previously (Kent and Mellor,

1995; Wang et al., 2011a). Yeast genomic DNAs were prepared with phenol-chloroform extraction

followed by ethanol precipitation. The DNA was then digested by EcoRI and separated on a 1.6%

agarose gel. Digestion patterns were analyzed by indirect-end-labeling. The [32P]dCTP incoropo-

rated probe whose sequence was listed in Supplementary file 3 was used for hybridization.

Preparation of yeast chromatin and chromatin immunoprecipitation(ChIP) assayYeast chromatin was prepared as described previously (Peng and Zhou, 2012). Specifically, mono-

nucleosomes were purified as described previously for detecting the level of H3N, H3K4me3,

H3K36me3 and H3K79me2/3. Chromatin was boiled for 10 min in SDS-PAGE loading buffer and




separated in 15% SDS-PAGE, and then subjected to western blotting. The chromatin immunoprecip-

itation (ChIP) assay was performed as described previously (Wang et al., 2011b).

Quantification of western blottingWe detected the linear range of all the antibodies. Then we loaded our samples in the linear range

and performed a western blot. Quantification of the western blot signals was carried out using

ImageJ software (RRID:SCR_003070).

Northern blot analysisTotal RNA was extracted using the Yeast RNA extraction kit (Qiagen), resolved on agarose-formal-

dehyde gels and transferred to Hybond-N+ membrane (GE). RNA was crosslinked to the membrane

by UV irradiation. Hybridization was carried out in 7% SDS, 1 mM sodium pyrophosphate, 1 M

Na2HPO4, 150 mM NaH2PO4, and 1 mM EDTA. Probes were generated by PCR.

RNA-Seq analysisThe method for constructing RNA-Seq libraries was modified from the TruSeq DNA sample prepara-

tion kit protocol (Illumina). Briefly, total RNA was isolated using the RNeasy midi kit (Qiagen). The

mRNA was purified from total RNA by Dynaloligo(dT) beads (Invitrogen, CA, USA). The first and sec-

ond strand cDNAs were synthesized using the SuperScript III CellsDirect cDNA Synthesis Kit (Invitro-

gen) and the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen), respectively. The

resulting double-stranded DNA was subjected to DNA repair and end-polishing (blunt-end)

using the End-It DNA End-Repair Kit (Epicentre). The DNA was then purified with the QIAquick PCR

Purification Kit (Qiagen) and a dA-tail was added using the 3’�5’ exo-Klenow Fragment (NEB). The

resulting purified fragments were ligated to adaptor oligo mix (Illumina) using Quick T4 DNA ligase

(NEB). The 200–500 bp ligation products were recovered from a 2% (w/v) agarose gel using

the Qiagen gel extraction kit and were PCR amplified with Illumina primers using the KAPA

HiFi HotStart kit. The 250–400 bp amplified products were purified again from a 2% agarose gel and

used directly for high-throughput sequencing. The raw paired-end reads contained the adapter

sequences: the P7 adapter (read1) is ’AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC’, the P5

adapter (read2) is ’AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT’. We used the FASTX Toolkit

(RRID:SCR_005534) to remove the adapter sequences. We trimmed the reads using TopHat (RRID:

SCR_013035), only mapping the reads to the transcriptome of sacCer3 (Apr. 2011) with the default

parameter. For the mapped reads, we then extracted the reads that have the ‘NH:i:1’ field. In order

to reduce the PCR duplicates’ bias, we kept the maximal three records at the same position.

To compare the gene expression profiles between WT (LHT001) and H3D/H3H strains, the aligned

reads were analyzed using Cuffdiff2 (RRID:SCR_001647) (Trapnell et al., 2012) to determine the

RPKM (Reads Per Kilobase per Million mapped reads) value for each sample. Genes with a change

greater than or equal to two folds and p-value � 0.001 were regarded as differentially expressed

genes and listed in Figure 2—source data 2. We identified 406 genes that were downregulated in

the H3D/H3H sample and 243 genes that were upregulated in the H3D/H3H sample compared with

the WT sample. We used FunSpec (RRID:SCR_006952, http://funspec.med.utoronto.ca/) to annotate

the differentially expressed genes to get the GO enrichment results (Robinson et al., 2002), which

were presented in Figure 2—source data 2.

For RNA-Seq analysis in glucose starvation experiments, we quantified the number of genes for

which at least one read was mapped (RPKM 6¼0). Fold changes in the transcription of genes under

glucose starvation, for genes listed in Figure 2—source data 2, were quantified as FCi,

j = log2((RPKM_1i,j/RPKM_1act1,j) /(RPKM_0i,j/RPKM_0act1,j)), where RPKM_1i,j and RPKM_0i,j refer to

RPKM for gene i in sample j after glucose starvation for 1 and 0 hr, respectively. We excluded the

gene i when p<0.05 (t-test) by comparing FCi,j in two independent experiments. For the remaining

genes, we calculated the average FCi,j (defined as FCai,j) of gene i in sample j using two replicates.

To evaluate whether the gene was potentially regulated by H3K4 methylation, we screened gene i

of which FCai;H3D=H3H is significantly different (p<0.05) from FCai;H3DK4R=H3HK4R and grouped it to set

DH_4 R4R. Genes in set DH_4 R4R were listed in Figure 8—source data 2.



https://scicrunch.org/resolver/SCR_003070





http://funspec.med.utoronto.ca/


Gene skewness score (GSS) modelThe skewness of gene transcript fold-change was defined by using the following model. If asymmet-

rically modified nucleosomes were involved in gene regulation, FCai;H3DK4R=H3H or FCai;H3D=H3HK4R

should prerequisitely fall between FCai;H3D=H3H and FCai;H3DK4R=H3HK4R. We therefore pooled gene i of

set DH_4 R4R into subset MID when Midi,j = 1. The value of Midi,j was calculated using the following

equation:

Midi;j ¼FCai;j�FCai;H3D=H3H

�

�

�

�þ FCai;j �FCai;H3DK4R=H3HK4R

�

�

�

�

FCai;H3D=H3H �FCai;H3DK4R=H3HK4R

�

�

�

�

The skewness score (GSS) of gene i in sample j was calculated by the equation:

GSSi;j ¼ log2FCai;j �FCai;H3DK4R=H3HK4R

�

�

�

�

FCai;j�FCai;H3D=H3H

�

�

�

�

; if Midi;j ¼ 1

Greater skewness of sample j to H3D/H3H leads to larger GSSi,j. Conversely, greater skewness of

sample j to H3DK4R/H3HK4R leads to smaller GSSi,j. The results of Midi,j and GSSi,j were listed in Fig-

ure 8—source data 3.

Gene type classificationThe gene i in set MID was classified into three subsets: (1) II, FCai;H3DK4R=H3H and FCai;H3D=H3HK4R

showing significant difference from both FCai;H3D=H3H and FCai;H3DK4R=H3HK4R. The maximum and mini-

mum values of GSS in this subset were defined as GSSmax and GSSmin, respectively; (2) III,

FCai;H3DK4R=H3H and FCai;H3D=H3HK4R exhibiting no difference from FCai;H3D=H3H but significant differ-

ence from FCai;H3DK4R=H3HK4R in the condition of each GSSi;H3DK4R=H3H and GSSi;H3D=H3HK4R larger than

GSSmax; and (3) I, FCai;H3DK4R=H3H and FCai;H3D=H3HK4R significantly differing from FCai;H3D=H3H rather

than FCai;H3DK4R=H3HK4R in the condition of each GSSi;H3DK4R=H3H and GSSi;H3D=H3HK4R smaller than

GSSmin. They were listed in Figure 8—source data 4.

StatisticsData were analyzed by Pearson’s product-moment test and Student t-test as indicated.

AcknowledgementsWe thank Drs Brian A Lenzmeier (Buena Vista University) and Hai Jiang for their critical reading of

the manuscript. This work was supported by grants from National Natural Science Foundation of

China (NSFC 31521061 and 31230040) and Ministry of Science and Technology (MOST

2016YFA0500701 to JQZ). The RNA-Seq data are available from GEO (Gene Expression Omnibus)

under accession number GSE104312 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=

GSE104312) and GSE88878 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE88878).

Additional information

Funding

Funder Grant reference number Author

National Natural ScienceFoundation of China

31521061 Jin-Qiu Zhou

National Natural ScienceFoundation of China

31230040 Jin-Qiu Zhou

Ministry of Science and Tech-nology of the People’s Repub-lic of China

2016YFA0500701 Jin-Qiu Zhou

The funders had no role in study design, data collection and interpretation, or the

decision to submit the work for publication.



http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE104312




Author contributions

Zhen Zhou, Conceptualization, Resources, Data curation, Software, Formal analysis, Investigation,

Methodology, Writing—review and editing; Yu-Ting Liu, Conceptualization, Resources, Data cura-

tion, Software, Formal analysis, Investigation, Methodology, Writing—original draft; Li Ma, Resour-

ces, Formal analysis, Methodology; Ting Gong, Ling-Li Zhang, Writing—review and editing,

Participated in discussion; Ya-Nan Hu, Resources, Methodology; Hong-Tao Li, Chen Cai, Resources,

Writing—review and editing; Gang Wei, Resources, Supervision, Methodology; Jin-Qiu Zhou, Super-

vision, Funding acquisition, Writing—review and editing

Author ORCIDs

Yu-Ting Liu, http://orcid.org/0000-0001-7501-7980

Jin-Qiu Zhou, http://orcid.org/0000-0003-1986-8611

Decision letter and Author response

Decision letter https://doi.org/10.7554/eLife.30178.034

Author response https://doi.org/10.7554/eLife.30178.035

Additional filesSupplementary files. Supplementary file 1. S. cerevisiae strains used in this study.


. Supplementary file 2. Antibodies used in this study.


. Supplementary file 3. Oligonucleotides used in this study.


. Transparent reporting form


Major datasets

The following datasets were generated:

Author(s) Year Dataset title Dataset URL

Database, license,and accessibilityinformation

Zhen Zhou, Li Ma,Ya-Nan Hu

2017 RNA transcription profile ofdifferent yeast mutants underglucose starvation (0.05% glucose)and comparison of transcriptome ofWT and H3D_H3H

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE104312

Publicly available atthe NCBI GeneExpression Omnibus(accession no.GSE104312)

Zhen Zhou, Li Ma,Yu-Ting Liu, Ya-NanHu

2017RNA transcription profile ofdifferent yeast mutants underglucose starvation (0.05% glucose)


Publicly available atthe NCBI GeneExpression Omnibus(accession no.GSE88878)

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Independent manipulation of histone H3 modifications in … · 2020. 4. 30. · bivalent nucleosome system that allowed for the constitutive assembly of asymmetrically modified sister